Neuroprotective activity of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (PAN-811), a cancer therapeutic agent

3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP) is a highly-hydrophobic small molecule that was originally developed for cancer therapy (Triapine, Vion Pharmaceuticals) due to its ability to inhibit ribonucleotide reductase, a key enzyme required for DNA synthesis. 3-AP has a high affinity for divalent cations, chelating the Fe(2+) at the R2 subunit of the enzyme and inhibiting formation of a tyrosyl radical essential for ribonucleotide reduction. We have demonstrated that 3-AP is also a potent neuroprotectant (as such, it is referred to as "PAN-811"). In vitro it completely blocks ischemic neurotoxicity at a concentration of 0.5 microM (EC(50) approximate, equals 0.35 microM) and hypoxic toxicity at 1.2 microM (EC(50) approximate, equals 0.75 microM). Full protection of primary cortical and striatal neurons can be achieved with 3-AP when it is added to the medium at up to six hours after an ischemic insult. 3-AP also suppresses cell death induced by neurotoxic agents, including staurosporine, veratridine and glutamate, indicating activity against a central target(s) in the neurodegenerative process. 3-AP acts via neutralization of two important intracellular effectors of excitatory neurotoxicity; calcium and free radicals. Its reported ability to elevate anti-apoptotic proteins is likely to be a consequence of the suppression of excessive intracellular free calcium. In a rat model of transient ischemia, a single bolus delivery of 3-AP 1 h after the initiation of ischemic attack reduced infarct volume by 59% when administered i.c.v. (50 mug per rat) and by 35% when administered i.v. (1 mg/kg). In Phase I clinical trials in cancer therapy 3-AP had no cardiovascular, CNS or other major adverse effects. Thus, 3-AP has a high potential for development as a novel, potent neuroprotectant for the treatment of neurodegenerative diseases.     

Pharmaceutical development of a parenteral lyophilised formulation of the investigational anticancer agent ES-285.HCl

The aim of this study was to design stable parenteral pharmaceutical final products containing 25 mg and 50 mg ES-285.HCl per dosage unit for use in phase I clinical trials. ES-285.HCl drug substance was fully characterised and showed very slight solubility in water. The development of the pharmaceutical product, containing 2-hydroxypropyl-beta-cyclodextrin, is discussed in view of formulation optimisation and manufacture. The developed freeze-dried products were found stable for at least 6 months at an accelerated storage condition of 25 +/- 2 degrees C/60 +/- 5% relative humidity and for at least 12 months at the designated long term storage condition of 5 +/- 3 degrees C, in the dark. Phase I trials using ES-285.HCl 25 mg/vial and 50 mg/vial final products are currently ongoing.     

A phase 2 consortium (P2C) trial of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP) for advanced adenocarcinoma of the pancreas

3-Aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, Triapine) is a novel small molecule inhibitor of ribonucleotide reductase (RR) with clinical signs of activity in pancreatic cancer. Therefore, the Phase 2 Consortium (P2C) initiated a trial (two single stage studies with planned interim analysis) of 3-AP at 96 mg/m(2) intravenously days 1-4 and 15-18 of a 28-day cycle in both chemotherapy-naive and gemcitabine-refractory (GR) patients with advanced pancreatic cancer. The primary endpoint was survival at six months (chemotherapy-naive) and four months (GR). Secondary endpoints were toxicity, response, overall survival, time to progression and mechanistic studies. Fifteen patients were enrolled including one chemotherapy-na?ve and 14 GR. The chemotherapy-na?ve patient progressed during cycle 1 with grade 3 and 4 toxicities. Of 14 GR patients, seven received two cycles, six received one cycle and one received eight cycles. Progression precluded further treatment in 11 GR patients. Additionally, one died of an ileus in cycle 1 considered related to treatment and two stopped treatment due to toxicity. Five GR patients had grade 4 toxicities possibly related to 3-AP and six GR patients had grade 3 fatigue. Toxicities and lack of meaningful clinical benefit prompted early study closure. Four-month survival in GR patients was 21% (95% CI: 8-58%). Correlative studies confirmed that 3-AP increased the percentage of S-phase buccal mucosal cells, the presence of multidrug resistance gene polymorphisms appeared to predict leukopenia, and baseline pancreatic tumor RR M2 expression was low relative to other tumors treated with 3-AP. In conclusion, this regimen appears inactive against predominantly GR pancreatic cancer. RR M2 protein may not have a critical role in the malignant potential of pancreatic cancer.     

A Phase II study of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP) and gemcitabine in advanced pancreatic carcinoma. A trial of the Princess Margaret Hospital Phase II consortium

3-Aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, Triapine, Vion Pharmaceuticals, New Haven, CT) is an inhibitor of the M2 subunit of ribonucleotide reductase (RR). Preclinical testing demonstrates synergy between 3-AP and gemcitabine. Phase I studies of the combination have suggested tolerability and some initial evidence of efficacy. Therefore, a phase II study of gemcitabine plus 3-AP in advanced pancreatic carcinoma was undertaken. In this two-step phase II trial, patients with advanced pancreatic adenocarcinoma who had not received prior chemotherapy for advanced disease were treated with 3-AP 105 mg/m(2) given over 2 h. Four hours after the 3-AP infusion was completed, gemcitabine 1,000 mg/m(2) was given over 30 min. Both drugs were given on days 1, 8 and 15 of a 28-day cycle.Twenty-six patients were enrolled to the study. One patient withdrew consent prior to receiving any treatment and is excluded from all further analyses. Four patients discontinued treatment due to adverse effects. Grade 3/4 hematological adverse events included neutropenia, thrombocytopenia, lymphopenia, leukopenia and anemia and the most frequent non-hematological adverse events were fatigue and pain. No objective responses were observed. Eleven patients had stable disease (SD). In five of these eleven patients, SD lasted for more than 6 months. The median time to progression was 4.1 months and the 6 month progression-free survival rate was 29%. The median survival was 9.0 months with a 1-year survival of 28.0%. The combination of 3-AP and gemcitabine is associated with moderate toxicity in patients with advanced pancreatic cancer. This two-stage trial was stopped after stage I due to lack of antitumour activity. On the basis of this clinical trial, the combination of gemcitabine and 3-AP at this dose and schedule does not warrant further study in this patient population.     

Development of an omeprazole parenteral formulation with hydroxypropyl-beta-cyclodextrin

Because of both the low solubility and stability of omeprazole in an aqueous environment, cyclodextrins (CDs) were added as inclusion complexation agents and stability enhancers in a parenteral formulation. Stability curves of omeprazole in different aqueous media were compared to determine which was most appropriate to prepare a formulation. The aimed preparation contains 40 mg omeprazole in an as low a volume of solvent as possible. A first assay in 40% hydroxypropyl-beta-cyclodextrin (HP-beta-CD) at pH 7.4 and 8.0 was not successful because degradation occurred before omeprazole was completely dissolved. Another manufacturing method was therefore tested. It concerned the use of an ammonia-based solvent in which omeprazole easily dissolved at high pH values, but a lower pH was reached after lyophilization. The inclusion capability of different cyclodextrins (CDs), suitable for parenteral use, was compared. The preparations that were finally selected (i.e., 40 mg omeprazole in 10 mL of a 40% HP-beta-CD ammonia/water-solution with an initial pH of 11 or 12, were found, after lyophilization, to be in agreement with the requirements for parenterals.     

Development and testing of an improved parenteral formulation of phenytoin using 2-hydroxypropyl-beta-cyclodextrin

The objective of this study was to increase the solubility of phenytoin by complexing it with varying concentrations of 2-hydroxypropyl-beta-cyclodextrin (HPBCD) and create an entirely aqueous formulation with a pH significantly closer to physiologic pH (7.4). The phenytoin-HPBCD complexation was characterized using phase-solubility analysis at HPBCD concentrations ranging from 10 to 50% w/v over the pH range of 7.4-11.0. The two most promising formulations, i.e., a formulation consisting of 40% HPBCD at pH 10.4, and a second formulation consisting of 20% HPBCD at pH 11.0, were selected for further study. Both formulations were entirely aqueous and had a significantly decreased pH compared to the original commercial formulation (Parke-Davis, pH 12.0). These formulations also exhibited a significantly decreased tendency to precipitate in vitro. The tissue irritation potential of the 20% w/v HPBCD formulation at pH 11.0 was found to be reduced considerably compared to the commercial injection in a BALB/c mouse model.     

Development and funding of a pharmacy-based investigational drug service

The development, implementation, and operation of a pharmacy-based investigational drug service (IDS) at a university medical center are described. Before the IDS was established, pharmacy participation in investigational drug research was limited to the preparation of novel dosage forms. Medication errors, improper storage and labeling, and inadequate inventories of investigational drugs were common problems. Stepped-up enforcement by FDA and the National Cancer Institute (NCI) of guidelines for investigational drug control prompted the formation of a multidisciplinary task force, which recommended that the department of pharmaceutical services expand its support of investigational drug studies to include inventory control, record keeping, and clinical services. The IDS is supported by both the hospital and the school of medicine and currently receives 36% of its funding from principal investigator grants and contracts. The IDS coordinates more than 100 study protocols and dispenses more than 4000 doses of investigational drugs annually. The IDS is staffed by 1.0 full-time equivalent (FTE) clinical pharmacist and 0.5 FTE technician. Inventory control and billing functions are performed by a departmental microcomputer system. The IDS has demonstrated a positive gross margin for each of its first two years of operation. Problems associated with the control and use of investigational drugs at this institution have been successfully corrected by the implementation of a pharmacy-based IDS.     

Synthesis and antitumor activity of amino derivatives of pyridine-2-carboxaldehyde thiosemicarbazone.

Various substituted pyridine-2-carboxaldehyde thiosemicarbazones (12 compounds) have been synthesized and evaluated for antineoplastic activity in mice bearing the L1210 leukemia. Oxidation of 3-nitro-2-picoline,5-nitro-2-picoline,3-nitro-2,4-lutidine, and 5-nitro-2,4-lutidine with selenium dioxide was employed to generate the corresponding pyridine-2-carboxaldehydes, which were then converted to cyclic ethylene acetals and subsequently reduced to amino and hydroxyamino derivatives by catalytic hydrogenation. Condensation of nitro aldehydes and acetals with thiosemicarbazide afforded the respective thiosemicarbazones. Acetylation of the amino acetals and alkylsulfonation of the 5-amino acetal, followed by condensation with thiosemicarbazide was employed to yield amide thiosemicarbazones. The most active compounds synthesized were 3-aminopyridine-2-carboxaldehyde thiosemicarbazone and 3-amino-4-methylpyridine-2-carboxaldehyde thiosemicarbazone which produced against the L1210 leukemia, % T/C values of 246 and 255, and 40% 60-day long-term survivors at two daily doses of 40 mg/kg and 10 mg/kg, respectively, for six consecutive days.     

Pharmaceutical development, quality control, stability and compatibility of a parenteral lyophilized formulation of the investigational polymer-conjugated platinum antineoplastic agent AP5346

AP5346 is a low molecular weight polymer-conjugated platinum antineoplastic agent. The lyophilized drug product has completed a phase I clinical trial. In order to guarantee a constant quality of AP5346 pharmaceutical products, quality control and analysis of the drug substance and final product were performed. The identity of AP5346 was confirmed using 1H NMR, 195Pt NMR and IR spectroscopy. Furthermore, the free platinum content, platinum release characteristics, molecular size and size distribution were established. With the selected analytical techniques, AP5346 could be distinguished very well from its polymeric analogues, such as AP5280 and AP5279. Stability experiments revealed that AP5346 final product is stable for 12 months at 5 degrees C, in the dark. For administration to patients, AP5346 final product is reconstituted with 5% w/v dextrose and diluted in infusion containers. To investigate the influence of container materials, the stability of AP5346 after reconstitution and dilution in infusion containers was determined. The infusion containers investigated were composed of glass, polyvinyl chloride (PVC, intraflex) and low density polyethylene (LD-PE, Ecoflac). AP5346 was shown to be stable after reconstitution and dilution with 5% w/v dextrose in these infusion containers for at least 96 h at 2-8 degrees C in the dark and at room temperature with ambient light conditions.     

Plasma protein binding of the investigational anticancer agent 2-methoxyestradiol

2-Methoxyestradiol (2ME2) is an endogenously produced metabolite of estradiol currently being tested in phase I and II clinical trials as an anticancer agent. Here, we examined the role of protein binding as a possible determinant of the pharmacokinetic behavior of 2ME2. The distribution of 2ME2 in plasma was studied in vitro using plasma from healthy human volunteers and ex vivo using plasma from patients with cancer receiving the drug orally. The equilibrium dialysis method used to characterize plasma protein binding of 2ME2 utilized a tracer amount of [H]-2-methoxyestradiol on a 96-well microdialysis plate with a 5-kDa cutoff membrane and 250 mul of plasma. The time to equilibrium was approximately 24 h and the mean unbound fraction of 2ME2 (fu) over the observed concentration range in plasma of patients receiving 2ME2 orally was 0.019+/-0.0043. The mean fu was 0.027+/-0.0019 in plasma of healthy human volunteers. The binding was concentration independent, indicating a low-affinity, possibly nonspecific and nonsaturable process. The binding was also unaffected by the presence of 2-methoxyestrone, one of the major metabolites of 2ME2. 2ME2 was found to bind in decreasing order to plasma>albumin>alpha1-acid glycoprotein>sex-hormone-binding globulin. Plasma concentration-time profiles of total 2ME2 and unbound 2ME2 concentrations in a patient with cancer receiving 2ME2 as a single oral dose were parallel to each other. Thus, indicating that plasma protein binding is not an important consideration in pharmacokinetic monitoring of 2ME2.     

Triapine (3-aminopyridine-2-carboxaldehyde- thiosemicarbazone): A potent inhibitor of ribonucleotide reductase activity with broad spectrum antitumor activity

Previous studies from our laboratories have shown that (a) Triapine() is a potent inhibitor of ribonucleotide reductase activity and (b) hydroxyurea-resistant L1210 leukemia cells are fully sensitive to Triapine. In an analogous manner, Triapine was similarly active against the wild-type and a hydroxyurea-resistant subline of the human KB nasopharyngeal carcinoma. Triapine was active in vivo against the L1210 leukemia over a broad range of dosages and was curative for some mice. This agent also caused pronounced inhibition of the growth of the murine M109 lung carcinoma and human A2780 ovarian carcinoma xenografts in mice. Optimum anticancer activity required twice daily dosing due to the duration of inhibition of DNA synthesis which lasted about 10 hr in L1210 cells treated with Triapine in vivo. DNA synthesis in normal mouse tissues (i.e. duodenum and bone marrow) uniformly recovered faster than that in L1210 leukemia cells, demonstrating a pharmacological basis for the therapeutic index of this agent. Triapine was more potent than hydroxyurea in inhibiting DNA synthesis in L1210 cells in vivo, and the effects of Triapine were more pronounced. In addition, the duration of the inhibition of DNA synthesis in leukemia cells from mice treated with Triapine was considerably longer than in those from animals treated with hydroxyurea. Combination of Triapine with various classes of agents that damage DNA (e.g. etoposide, cisplatin, doxorubicin, and 1-acetyl-1,2-bis(methylsulfonyl)-2-(2-chloroethyl)hydrazine) resulted in synergistic inhibition of the L1210 leukemia, producing long-term survivors of tumor-bearing mice treated with several dosage levels of the combinations, whereas no enhancement of survival was found when Triapine was combined with gemcitabine or cytosine arabinoside. The findings demonstrate the superiority of Triapine over hydroxyurea as an anticancer agent and further suggest that prevention by Triapine of repair of DNA lesions created by agents that damage DNA may result in efficacious drug combinations for the treatment of cancer.     

Approaches to reducing toxicity of parenteral anticancer drug formulations using cyclodextrins

Mitomycin C (MMC) is a clinically useful anticancer drug which can cause severe dermatological problems upon injection. It can cause delayed erythema and/or ulceration occurring either at or distant from the injection site for weeks or even months after administration. In an attempt to reduce the skin necrosis, complexation of MMC with cyclodextrins was studied in order to help increase patient compliance and acceptance. The complexation of MMC with 2-Hydroxypropylbetacyclodextrin (HPBCD) in the presence and absence of mannitol was studied and it was found that the mannitol present in the commercial formulation caused an increase in the binding of MMC to HPBCD. Isotonicity adjustment of hypotonic MMC formulations by the addition of normal saline did not change the degree of complexation with MMC. The complexed formulations were then tested to determine their antitumor efficacy using the B-16 melanoma cell model. No difference in antitumor activity between the complexed and uncomplexed MMC formulations was observed. Different MMC formulations were tested for their potential to produce skin irritation and/or toxicity using intradermal injections in a BALB/c mouse model in order to find the most suitable formulation. The skin ulceration studies indicated that there were no significant differences between the isotonic MMC solution and isotonic formulations of MMC complexed with HPBCD.     

Solubilization and stabilization of an investigational antineoplastic drug (NSC no. 278214) in an intravenous formulation using an emulsion vehicle

The use of parenteral fat emulsions for development of an extemporaneous preparation of an intravenous formulation of a poorly water-soluble and unstable investigational anticancer agent (NSC no. 278214) is presented. The incorporation into a commercial fat emulsion of the drug, dissolved in dimethylacetamide-cremophor solution, results in a suitable parenteral formulation in which the drug is approximately 100-fold more stable than in simple aqueous solutions.     

Synthesis, antimicrobial and anticancer activity of new thiosemicarbazone derivatives

Thiosemicarbazones of p-aminobenzoic acid (PABA) were synthesized and tested for their antimicrobial and anticancer activity. Hydroxamate derivatives 4a-4l were found to have better antimicrobial and anticancer activity than their acid counterpart. Compound 4d was found to have good antimicrobial activity against Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Vibrio cholerae, and Bacillus subtilis with IC(50) value of about 1 μM. Compound 4f showed potent antifungal activity against Candida albicans (IC(50) = 1.29 μM) and compound 4h showed potent anticancer activity (IC(50) = 0.07 μM).     

抗肿瘤药物吉非替尼的研究进展

吉非替尼(gefitinib,Iressa,ZD1839)是一种口服选择性表皮生长因子受体酪氨酸激酶抑制剂.临床前研究发现它能抑制多种类型肿瘤细胞的生长,临床Ⅰ、Ⅱ期研究表明在晚期非小细胞肺癌(NSCLC)患者中,吉非替尼单独应用显示出明显的抗瘤活性并使症状减轻.2003年5月FDA批准其上市.综述了吉非替尼的分子作用机制、体内外抗肿瘤作用以及临床试验等方面的最新进展.     

Photoinduced particulate matter in a parenteral formulation for bisnafide, an experimental antitumor agent.

This paper assesses the cause of particulate formation in vials of the experimental antitumor agent bisnafide and investigates pharmaceutical techniques to reduce the number of particulates in the product. Solution preparation and particulate isolation were performed under Class 100 laminar air flow. Reversed-phase HPLC and infrared microscopy were used to characterize drug and isolated particulate matter, whereas a Hiac particle counter was used to quantify the particulate matter. Particulate matter was observed following agitation of the drug solutions and was found to be associated with specific lots of drug substance. HPLC of the isolated particulate matter indicated that the particulates consisted largely of bisnafide and impurities that were identified as the products of photodegradation, confirmed to be the result of the photolytic cleavage of bisnafide to form a poorly soluble aldehyde. The aldehyde may, in turn, interact with bisnafide molecules to form the particulate matter as suggested by the observed pH-dependent reversibility of the particulate phenomenon. The particulate matter could be reduced by protecting solutions of bisnafide from light during chemical synthesis and production of the dosage form and, alternatively, by reducing the solution pH to 3.0 or less, addition of surfactants below their critical micelle concentration, and removal of impurities by froth flotation of the bisnafide solutions.     

Development of parenteral formulation for a novel angiogenesis inhibitor, CKD-732 through complexation with hydroxypropyl-beta-cyclodextrin

The effect of hydroxypropyl-beta-cyclodextrin (HP-beta-CyD) on the aqueous solubility and chemical stability of O-(4-Dimethylaminoethoxycinnamoyl)fumagillol (CKD-732), a new angiogenesis inhibitor, was investigated with an aim of preparing a stable and effective parenteral formulation. The CKD-732/HP-beta-CyD inclusion complex was obtained in solid state by freeze-drying and characterized in solution by proton nuclear magnetic resonance (1H NMR). Then, the pharmacokinetic profile in rats and the in vivo tumor growth inhibitory activity in mice following the parenteral administration of aqueous CKD-732/HP-beta-CyD complex were compared to those of CKD-732.hemioxalate solution having an equivalent concentration. The aqueous solubility of CKD-732 was markedly increased by the combination of pH adjustment and HP-beta-CyD complexation through a soluble 1:1 inclusion complex formation, which was supported by NMR spectroscopy. The hydrolysis of CKD-732 following pseudo first-order kinetics was decelerated moderately but significantly in acidic and basic solutions in the presence of HP-beta-CyD. The stability of lyophilized CKD-732/HP-beta-CyD complex was also drastically improved after storage in various conditions. The intravenous pharmacokinetic profile and the subcutaneous in vivo tumor growth inhibitory activity of aqueous CKD-732/HP-beta-CyD complex were not significantly different from those of CKD-732.hemioxalate solution with the favorable reduction of irritation. These results demonstrate that the CKD-732/HP-beta-CyD complex is an attractive formulation for use in the parenteral delivery of CKD-732.     

Physicochemical characterization and an injection formulation study of water insoluble ZCVI4-2, a novel NO-donor anticancer compound

ZCVI(4)-2 was a novel nitric oxide-releasing glycosyl derivative of oleanolic acid that displayed strong cytotoxicity selectively against human hepatocellular carcinoma in vitro and in vivo. In this study, ZCVI(4)-2 was characterized by FT-IR spectroscopy, differential scanning calorimetry, powder X-ray diffractometry, Raman spectroscopy, hygroscopicity and stability. A high performance liquid chromatography method was also established for the quantitative determination of solubility and additional stability profile of ZCVI(4)-2. ZCVI(4)-2 was found to be an amorphous and stable solid with low solubility of less than 10 μg/mL. Based on the solubilization tests that included methods of cosolvency and micellization, the solution mixture of 5% Solutol HS-15, 5% 1, 2-propylene glycol and 5% anhydrous ethanol was determined to be the system for the preparation of the ZCVI(4)-2 early injection solution. The effect of pH, temperature, light and injectable isotonic glucose or NaCl solution on ZCVI(4)-2 injection was also investigated. Good stability was observed at all testing conditions. Under the conditions studied, the NO-releasing rate and amount of ZCVI(4)-2 from the early injection solution in rat plasma demonstrated a promising therapeutic efficacy while maintaining a good safety profile.